Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair–associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.

聚（ADP-核糖）聚合酶（PARP）抑制和BRCA之间的合成致死率缺陷被用来治疗乳腺和卵巢肿瘤。然而，对 PARP 抑制剂 (PARPis) 的耐药性很常见。为了确定潜在的耐药机制，我们在 BRCA2 缺陷小鼠胚胎干细胞中进行了全基因组 RNAi 筛选，并在 KB2P1.21 小鼠乳腺肿瘤细胞中进行了验证。我们发现对多种 PARPi 的抗性随着 TET2（十一十一易位）的表达降低而出现，TET2 通过将 5-甲基胞嘧啶 (5mC) 氧化为 5-羟基甲基胞嘧啶 (5hmC) 和其他产物来促进 DNA 去甲基化。BRCA2 缺陷细胞中的 TET2 敲低保护停滞的复制叉 (RF)。增加 5hmC 丰度通过招募碱基切除修复相关的无嘌呤/无嘧啶核酸内切酶 APE1，诱导 KB2P1.21 和人类癌细胞中停滞的 RFs 降解，独立于BRCA2状态。TET2 丢失不影响修复蛋白 RAD51 向双链断裂 (DSB) 位点的募集或与 RF 完整性相关的蛋白质丰度。TET2、其产物 5hmC 和 APE1 向停滞的 RF 募集的丧失促进了对化疗顺铂的抗性。我们的研究结果揭示了表观遗传标记 5hmC 在维持停滞 RF 完整性方面的先前未知作用以及对 PARPi 和顺铂的潜在耐药机制。